Receptor tyrosine kinases (RTKs) are cell surface proteins which, upon activation, signal to the cell interior to promote growth and differentiation. Elevated expression and aberrant signaling of RTKs is commonly found in tumor cells. Pharmaceutical inhibitors have been developed to inhibit the activity of RTKs but current drugs provide a benefit to a very low percentage of patients. Genetic variability between patients and compensatory signaling from other receptors are believed to be two of the main reasons behind resistance to the inhibitors. However, since we have not fully explored the mechanisms of RTK regulation our understanding of drug resistance and our ability to generate better inhibitors is limited. RTK signaling is terminated by ligand-induced internalization and receptor degradation in the lysosome (termed down-regulation). This process is heavily dependent upon receptor ubiquitylation. Following internalization, ubiquitylated receptors arrive at the sorting endosome, a compartment responsible for sending receptors to the lysosome for degradation or recycling them back to the plasma membrane. Proteins which block lysosomal targeting by promoting receptor recycling are considered negative regulators of receptor down-regulation. Inhibiting their function will promote receptor down-regulation, making them excellent targets for therapeutic inhibition. Our lab is investigating RTK down-regulation by studying the prototypical RTK, epidermal growth factor (EGF) receptor (EGFR). The main goal of this proposal is to identify new negative regulators of EGFR down-regulation. We have recently developed a novel screen which measures RTK down-regulation in response to various inhibitors. Using a library of highly-efficient short interfering RNA duplexes, which target over 100 ubiquitin-related genes we have identified a number of novel genes whose knockdown promotes EGFR down-regulation. This proposal will investigate in detail how two of these proteins control EGFR down-regulation. We will determine the subcellular localization of these proteins, particularly in relation to internalized EGFR. We will analyze how the conserved domains of these proteins function with ubiquitin to control cellular levels and signaling of EGFR. Furthermore, we will investigate the ability of the corresponding proteins to control cellular levels of other RTKs. Relevance - Tumor formation is often the result of abnormal cell growth caused by aberrant signaling from defective proteins. Current research, including the studies proposed here, is focusing on promoting the degradation of these defective proteins through normal cellular processes. [unreadable] [unreadable] [unreadable]